High-throughput screening (HTS) is a major method in early drug discovery and has yet to be fully applied to all human lipoxygenase (hLO) isozymes, critical enzymes in the progression of a number of human diseases. We have currently discovered over 20 novel inhibitors against human lipoxygenase primarily utilizing manual screening but have recently developed a high-throughput assay to increase the rate of discovery. We would now like to apply this HTS technology to the approximately 73,000 compounds at the NIH DPISMR to identify specific inhibitors to each of our three LO isozymes (12- hLO, 15-hLO-1 and 15-hLO-2). The specific inhibitors we discover will then be screened against specific cell lines to determine their in-vivo activity against prostate cancer cells and neuronal cells (as ischemia models). In parallel with the HTS, and at no extra cost to the NIH, we will dock the full HTS library against our three lipoxygenase homology models (12-hLO, 15-hLO-1 and 15-hLO-2) and compare the docking hits to those found by the HTS assay. This comparison will allow us to both improve our in silico methods and help us structurally interpret the HTS data we will obtain. Currently, we have shown both the accuracy and the reliability of this screen against a 47 compound flavonoid library and the NCI 3104 compound Mech./Div./Nat.Prod. library. In addition, Anton Simeonov, at NIH Chemical Genomics Center (NCGC), has successfully screened the LOPAC library with the Kalypsys robot and shown known LO inhibitors can be accurately found. Finally, modeling predictions with Prof. Matt Jacobson have already produced inhibitors from earlier screens. All of this data suggests that these aims are not only feasible but highly likely to be successful. Lipoxygenases (LO) are widely distributed throughout the plant and animal kingdoms and play a central role in the biology of these organisms. In plants they are involved in germination and senescence. In human tissue, there are three major human lipoxygenases, 5-, 12-, and 15-LO, whose primary enzymatic difference lies in their positional specificity on arachidonic acid (AA). The products of lipoxygenase are the precursors of hormones, such as leukotrienes and lipoxins, which have been implicated as critical signaling molecules in a variety of inflammatory diseases and cancers. There is currently strong evidence that if scientists can discover potent inhibitors against each specific lipoxygenase isozyme, these may become effective cellular probes and therapeutics against LO. This grant submission proposes to do exactly this for platlet 12-LO, reticulocyte 15-LO-1 and epidermal 15- LO-2. [unreadable] [unreadable] [unreadable]